Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14

Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomyces cerevisiae, The degree of in vivo phosphorylation of hexo kinase 2 at serine-14 is inversely related to the extracellular glucose con centration [Vojtek, A. B., and Fraenkel, D. G. (1990) fur. J, Biochem. 190, 371-375]; however, a physiological role of the modification causing the di ssociation of the dimeric enzyme in vitro [as effected by a serine-glutamat e exchange at position 14; Behlke et al. (1998) Biochemistry 37, 11989-1199 5] is unclear. This paper describes a comparative stopped-flow kinetic and sedimentation equilibrium analysis performed with native unphosphorylated h exokinase 2 and a permanently pseudophosphorylated glutamate-14 mutant enzy me to determine the functional consequences of phosphorylation-induced enzy me dissociation, The use of a dye-linked hexokinase assay monitoring proton generation allowed the investigation of the kinetics of glucose phosphoryl ation over a wide range of enzyme concentrations. The kinetic data indicate d that monomeric hexokinase represents the high-affinity form of isoenzyme 2 for both glycolytic substrates. Inhibition of glucose phosphorylation by ATP [Moreno et al, (1986) Eur, J, Biochem. 161, 565-569] was only observed at a low enzyme concentration, whereas no inhibition was detected at the hi gh concentration of hexokinase 2 presumed to occur in the cell. Pseudophosp horylation by glutamate substitution for serine-14 increased substrate affi nity at high enzyme concentration and stimulated the autophosphorylation of isoenzyme 2, The possible role of hexokinase 2 in vivo phosphorylation at serine-14 in glucose signaling is discussed.